JP7242042B2 - Osteoclast differentiation inhibitor and internal medicine or food and drink composition for prevention, treatment and improvement of bone resorption disease - Google Patents

Description

The present invention relates to oral preparations and food and drink compositions that contain 4'-demethyl nobiletin as an active ingredient and have osteoclast differentiation inhibitory action and are effective for bone resorption diseases.

Osteocytes include osteoblasts, which form bones, and osteoclasts, which destroy bones, and bones are reconstructed through the constant repetition of the actions of these cells. However, in osteoporosis, these balances are disturbed, and the amount of bone resorption by osteoclasts relatively exceeds the amount of bone formation, resulting in a decrease in bone mass, leading to the onset of osteoporosis (see, for example, Non-Patent Document 1). . Osteoclasts are differentiated from monocyte/macrophage progenitor cells, and macrophage cells present in bone are considered to be osteoclast progenitor cells (see, for example, Non-Patent Document 2). Alveolar bone resorption in periodontal disease is also reported to be due to activation of osteoclasts (see, for example, Non-Patent Document 3). Therefore, it is considered that ingredients having osteoclast differentiation inhibitory action are effective for bone resorption diseases such as osteoporosis and periodontal disease.

On the other hand, oral bisphosphonate preparations and calcitonin preparations are used as drugs that suppress bone resorption. Therefore, side effects such as shock symptoms may occur. Since osteoporosis therapeutic drugs require long-term administration, their safety is particularly strongly demanded. Under such circumstances, among food materials that can be taken on a daily basis, some ingredients that have osteoclast differentiation inhibitory action and are effective in the treatment of osteoporosis have been reported (see, for example, Patent Documents 1 and 2). . However, these have not been sufficiently effective.

Wu Ji Tai et al., Bulletin of Research Institute for Biological Functions, No. 4, p.11-14 (2004) Naoyuki Takahashi, Journal of the Japanese Orthopedic Society, Vol.88, p.855-859 (2014) Michihiko Usui et al., Journal of the Japanese Society of Periodontology, Vol.57, p.120-125 (2015) Masahiro Urade, Journal of the Japanese Society of Oral and Maxillofacial Surgery, Vol.56, No.5, p.292-297 (2010)

Japanese Patent Application Laid-Open No. 2009-215250 Japanese Patent Application Laid-Open No. 2009-107995

To solve the above problems, the present invention provides an oral preparation that has an excellent osteoclast differentiation inhibitory action among safe food materials and is effective for bone resorption diseases such as alveolar bone resorption in osteoporosis and periodontal disease. The purpose is to develop food and drink compositions.

Nobiletin, which is a polymethoxyflavonoid, is a flavonoid unique to citrus, and in recent years, various physiological effects such as cancer prevention, anti-aging, and anti-arteriosclerosis effects have come to be known.

The present inventors have clarified that the main component nobiletin is converted to 4'-demethyl nobiletin by fermenting citrus peels containing a large amount of nobiletin with a specific type of Aspergillus oryzae. The present inventors also found that 4'-demethyl nobiletin has an excellent memory improving action (see Japanese Patent No. 5667561). The present inventors further investigated the functionality of 4'-demethyl nobiletin and found that it has a strong osteoclast differentiation inhibitory effect.

That is, the present invention relates to an osteoclast differentiation inhibitor containing 4'-demethyl nobiletin as an active ingredient.
The present invention also relates to food and drink compositions for suppressing osteoclast differentiation containing 4'-demethyl nobiletin as an active ingredient.
The present invention also relates to an oral preparation for preventing or treating bone resorption diseases containing 4'-demethyl nobiletin as an active ingredient.
The present invention also relates to food and drink compositions for preventing or improving bone resorption diseases, containing 4'-demethyl nobiletin as an active ingredient.

INDUSTRIAL APPLICABILITY The present invention makes it possible to provide oral preparations and food and beverage compositions that contain 4'-demethyl nobiletin as an active ingredient and are effective for bone resorption diseases such as alveolar bone resorption in osteoporosis and periodontal disease. and

In addition, since 4'-demethyl nobiletin is a safe food-derived ingredient obtained from the fermented product of citrus peel aspergillus oryzae, the oral preparation and the food and drink composition of the present invention are well-experienced and have no side effects. .

Fig. 2 is a microscopic image showing osteoclast differentiation induction by addition of sRANKL. (Example 3) In the figure, the left shows the results of the negative control (no addition), and the right shows the results of the addition of sRANKL at 300 ng/ml. Fig. 2 is a microscopic image showing the osteoclast differentiation inhibitory effect of adding a 4'-demethyl nobiletin isolate. (Example 3) In the figure, the upper left shows the results of the negative control (no addition), the upper right shows the results of the addition of sRANKL at 300 ng/ml, the lower left shows the results of sRANKL + 4'-demethyl nobiletin, and the lower right shows the results of sRANKL + nobiletin. Fig. 3 is a graph showing the osteoclast differentiation inhibitory effect of the addition of 4'-demethyl nobiletin isolate. (Example 3) In the figure, the vertical axis indicates the ratio (%) of osteoclast differentiation, and the bar graphs are, from the left, negative control (no addition), addition of sRANKL, addition of 5, 10, and 20 µM 4'-demethyl nobiletin, The results for 5, 10 and 20 µM nobiletin addition are shown, and the bar indicates the standard deviation. ** indicates a significant difference (p<0.01) from the nobiletin-added group at the same concentration. Fig. 3 is a graph showing the osteoclast differentiation inhibitory effect of adding 4'-demethyltangeretin isolate. (Example 3) In the figure, the vertical axis indicates the ratio (%) of osteoclast differentiation, and the bar graphs are negative control (no addition), sRANKL added, 4'-demethyltangeretin added at 5, 10, and 20 µM from the left. , tangeretin added at 5, 10 and 20 μM, and the bar indicates the standard deviation. ** indicates a significant difference (p<0.01) with respect to the same concentration of tangeretin-added group, and * indicates a significant difference (p<0.05) with respect to the same concentration of tangeretin-added group. Fig. 2 is a microscopic image showing osteoclast differentiation inhibitory action of a composition containing 4'-demethyl nobiletin. (Example 3) In the figure, the upper left is the negative control (no addition), the upper right is the addition of 300 ng/ml of sRANKL, the lower left is the addition of 10 μg/ml of the composition containing sRANKL + 4'-demethyl nobiletin, and the lower right is the addition of sRANKL + 4'-demethyl nobiletin. The results of adding 20 μg/ml of the containing composition are shown.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail.
An embodiment of the present invention relates to an osteoclast differentiation inhibitor containing 4′-demethyl nobiletin as an active ingredient.
Another embodiment of the present invention relates to an oral preparation for prevention, treatment or improvement of bone resorption diseases, and food and drink compositions used for these purposes.

[4'-demethyl nobiletin]
4′-Demethyl nobiletin (Formula 1) is not commercially available, but a synthetic product can be used. Alternatively, 4'-demethyl nobiletin pure product (isolated) or 4'-demethyl nobiletin obtained by aspergillus fermentation using nobiletin-containing citrus fruits, especially pericarp, by the method described in previously reported Japanese Patent No. 5667561 A '-demethyl nobiletin-containing composition can be used. The method described in Japanese Patent No. 5667561, which utilizes biotransformation of nobiletin by fermentation with Aspergillus oryzae, is simpler and less expensive than the synthetic product, and is thus preferred.

4'-demethyl nobiletin is also one of the metabolites formed after nobiletin is absorbed in the body. Citrus peel is used as a raw material for sweets such as marmalade and candied, and mandarin oranges (Citrus reticulata) with a high nobiletin content have been eaten as chimps for many years. can be safely taken orally without worrying about side effects.

A method for producing 4'-demethyl nobiletin and a composition containing the same according to the method described in Japanese Patent No. 5667561 will be described below.

[Fermentation raw material]
The raw materials for koji mold fermentation are citrus 'fruits' (whole fruits including peel, juice, pulp, seeds, etc.) containing the polymethoxyflavonoid nobiletin. From a utilization point of view, it is desirable to use the 'pericarp'.

In addition, as the type of citrus, any variety and strain (for example, Ponkan, Shikwasha, Tangerine, Tachibana, etc.) can be used as long as it is a citrus containing nobiletin.

As the raw material for fermentation, those containing other parts of the citrus plant body (e.g., leaves, buds, stems, flowers, etc.) may be used, but those containing no nobiletin do not contain these parts. is desirable.

The above citrus fruits are preferably harvested or harvested fresh or washed with water, but dried, frozen, or long-term preserved citrus fruits can also be used. Although citrus fruits may be used as they are, it is desirable to subject them to at least one crushing treatment, such as chopping, crushing, or crushing.

The crushing process includes a wide range of actions, such as chopping the citrus into large pieces, chopping into small pieces, crushing, grinding, pulverizing, and the like. Preferably, it can be carried out by chopping into large pieces of about 1 to several centimeters.

Furthermore, an extract (extract, dried product) obtained by extracting nobiletin in advance from the fermentation raw material, or a product isolated as pure nobiletin can be used for fermentation. In addition, it is preferable that the raw material for fermentation is heat-treated to kill various bacteria in the raw material before performing the koji mold fermentation described later.

[Aspergillus fermentation]
Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, and Aspergillus sojae are examples of the koji mold for fermenting the fermentation raw material. , Aspergillus saitoi, Aspergillus usamii, Rhizopus filamentous fungi (also known as Rhizopus fungi), and the like can be used. Moreover, you may mix and use these.

Among the koji molds, Aspergillus kawachii, Aspergillus awamori, Aspergillus oryzae, and Aspergillus niger are preferably used to produce 4'-demethyl nobiletin. It can be obtained with a high content.

As a method of inoculating the fermented raw material with the koji mold, the spores of the koji mold can be directly sprinkled onto the fermented raw material so that the spores adhere to the fermented raw material. Alternatively, a medium obtained by preliminarily fermenting the koji mold by liquid culture may be inoculated so as to spread over the entire fermentation raw material.

As for the fermentation conditions when the koji mold is inoculated into the raw materials for fermentation, it is desirable to carry out the fermentation under aerobic conditions. It is preferable to spread the fermentation raw material evenly over the bottom of such a container so that the contact area with the air is large.

The fermentation temperature is preferably 10 to 40°C, more preferably 20 to 40°C, and still more preferably 25 to 32°C, as suitable conditions for growing the koji mold. In addition, it is preferable to ferment in a dark place as conditions suitable for the growth of the koji mold. Moreover, it is preferable that the raw material contains sufficient moisture.

The fermentation period for obtaining a large amount of 4'-demethyl nobiletin is preferably 2 to 21 days, more preferably 3 to 14 days, still more preferably 4 to 12 days. If the fermentation period is less than 2 days, the fermentation by the koji mold will hardly proceed, and sufficient 4'-demethyl nobiletin will not be obtained. On the other hand, if it exceeds 21 days, the decomposition of 4'-demethyl nobiletin produced by microbial conversion progresses, and the favorable citrus-derived aroma disappears.

In the koji mold fermentation, nobiletin is demethylated by an enzyme secreted from the koji mold and converted to 4'-demethyl nobiletin. Therefore, instead of performing Aspergillus oryzae fermentation, solution extraction is performed from the Aspergillus oryzae or the fermented product obtained after fermentation to obtain an enzyme solution containing an enzyme that demethylates nobiletin, and the enzyme is used to perform an enzymatic reaction with the raw material. It is also possible to obtain 4'-demethyl nobiletin by obtaining reactants. Specifically, an enzymatic reaction can be performed by recovering a water-dissolved product from the fermented product after the koji mold fermentation and using it as a crude enzyme solution.

By carrying out the above koji mold fermentation, all of the polymethoxyflavonoid nobiletin contained in the fermentation raw material is converted to 4'-demethyl nobiletin. Specifically, by fermenting the raw material aspergillus oryzae, 4'-demethyl nobiletin has a high content of about 0.5 to 1.5 mass% (specifically, about 1 mass%) per dry weight. Aspergillus oryzae fermented product can be obtained. Therefore, the koji mold fermented product obtained here can be used as it is or processed (for example, minced, crushed, pulverized, dried, etc.) to obtain osteoclasts of the present embodiment. It can be used as an active ingredient of differentiation inhibitors, internal medicines and food and drink compositions.

[Solution extraction]
In view of the purity, in the production of the osteoclast differentiation inhibitor, oral preparation and food and drink composition of the present embodiment, solution extraction is performed from the fermented product obtained after the koji mold fermentation to obtain an extract. It is desirable to obtain

The solution extraction can be performed directly on the fermented product of Aspergillus oryzae. It is desirable to perform solution extraction on the obtained material.

Solvents used for solution extraction can be water, buffer solutions, organic solvents, or these hydrous solvents. Examples of organic solvents include lower aliphatic alcohols such as ethanol, methanol, isopropanol and butanol, acetone, ethyl acetate and chloroform. Among these solvents, water, ethanol, and hydrous ethanol are particularly preferred in terms of extraction efficiency, ease of handling, and safety. In particular, by performing extraction using hydrous ethanol having a final concentration of 55% or more, preferably a final concentration of 60% or more, more preferably a final concentration of 80% or more (both v/v), It is preferable because elution of sugars can be suppressed and the extraction efficiency of 4'-demethyl nobiletin can be improved.

As the extraction conditions, the solvent is added in an amount of 1 to 50 times, preferably 2 to 15 times (both mass ratios) with respect to the raw material (preferably fragmented material), and the temperature is from 0 ° C. to the boiling point of the solvent. It is possible to extract 4'-demethyl nobiletin by soaking or shaking under conditions, preferably room temperature to the boiling point of the solvent or less, for 5 minutes to 1 month, preferably 20 minutes to 1 week. be. The obtained extract can be concentrated and dried by freeze-drying or drying using an evaporator or the like.

Solution extraction can also be performed multiple times with different solvents. For example, after the first extraction is performed with a low-concentration solvent such as water or low-concentration hydrous alcohol, a second extraction is performed using a solvent with a higher concentration than the previous solvent to obtain 4'-demethyl nobiletin. can improve the extraction efficiency of

The extract obtained as described above (the extract or the concentrated dried product) has an excellent osteoclast differentiation inhibitory action and an action to prevent, treat or improve bone resorption diseases such as osteoporosis and alveolar bone resorption in periodontal disease. Therefore, it can be used as it is as an active ingredient of the osteoclast differentiation inhibitor, internal medicine, or food and drink composition of the present embodiment.

〔purification〕
In addition, the 4'-demethyl nobiletin content can be further increased by further purifying the above extract. Purification means include, for example, liquid-liquid separation extraction, column purification using silica gel, chemically modified silica gel, activated carbon, synthetic adsorption resin carrier, and the like. An example of suitable purification conditions when solution extraction is performed using hydrous ethanol is shown below as an embodiment.

First, ethanol is removed from the extract, and the resulting ethanol-free solution is applied to a column of porous synthetic adsorption resin (eg, Diaion HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.)) equilibrated with water. After removing the components eluted with water, the liquid eluted with 30 to 35% (v/v) ethanol is removed. Next, by collecting the components eluted with 44-46% (v/v) ethanol, a 4'-demethyl nobiletin-rich composition can be obtained. A composition containing 15% (w/w) or more of 4'-demethylnobiletin at a high content can be obtained by performing extraction and purification under the suitable conditions described above.

In addition, the 4'-demethyl nobiletin-containing composition obtained as described above is further subjected to ODS column chromatography (e.g., 45% methanol elution), thin layer chromatography (TLC) (e.g., hexane/ethanol (7:3) ), ODS-HPLC (for example, a mixed solvent of 37% (v/v) acetonitrile/water), etc., and the target peak can be collected to isolate pure 4'-demethyl nobiletin.

The 4'-demethyl nobiletin obtained above is a monodemethyl form of nobiletin in which the 4' position is demethylated. 4'-demethyl nobiletin becomes more polar due to demethylation, and has better solubility in alcohol and water than nobiletin. In addition, 4'-demethyl nobiletin has an osteoclast differentiation inhibitory effect superior to that of nobiletin, and is effective for bone resorption diseases such as alveolar bone resorption in osteoporosis and periodontal disease.

[Osteoclast differentiation inhibitor, internal medicine, and food and drink composition]
The osteoclast differentiation inhibitor of the present embodiment is characterized by containing 4'-demethyl nobiletin as an active ingredient.
The oral preparation of the present embodiment contains 4'-demethyl nobiletin as an active ingredient and is used for prevention or treatment of bone resorption diseases.
The food and drink composition of the present embodiment contains 4'-demethyl nobiletin as an active ingredient, has osteoclast differentiation inhibitory activity, and is used for prevention or improvement of bone resorption diseases.

The above 4'-demethyl nobiletin is obtained from each composition obtained by the above method ('composition directly containing fermentation product', 'solution extract', 'high content composition', 'purified product', etc.) The osteoclast differentiation inhibitor, internal medicine, and food/drink composition of the present embodiment can be produced by mixing the or isolate with various raw materials as an active ingredient. The term “oral medicine” includes pharmaceuticals and quasi-drugs. In addition, "food and drink compositions" include general food and drink, functional foods, and functional beverages.

The effective dose of 4'-demethyl nobiletin when orally ingesting the osteoclast differentiation inhibitor, oral preparation, or food and drink composition of the present embodiment is 1 mg or more, preferably 1 mg or more per day for an adult weighing 60 kg. By oral ingestion of 5 mg or more, excellent osteoclast differentiation inhibitory action and excellent preventive, therapeutic, and ameliorative action against bone resorption diseases can be obtained. Therefore, by ingesting the osteoclast differentiation inhibitor, oral preparation, or food and drink composition of the present invention in a form and ingestion method (number of times, amount) that can ensure this necessary amount, the above pharmacological action can be obtained. Be expected. However, it is desirable to appropriately determine the intake amount according to the subject's age, body weight, symptoms, intake schedule, preparation form, and the like.

In addition, the content of 4'-demethyl nobiletin in osteoclast differentiation inhibitors, internal medicines, and food and drink compositions may be any content as long as it is contained so as to ensure the above-mentioned necessary intake. can be contained so as to be 0.001% by mass or more, preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. Moreover, as an upper limit, 20 mass % or less can be mentioned.

Osteoclast differentiation inhibitors and internal preparations may be in the form of, for example, powder, fine granules, granules, toothpaste, etc. In addition to the form of filling these in capsules, they may be in the form of a liquid dispersed in water. form, cream form, tablet form obtained by mixing with excipients and the like.

In addition to 4'-demethyl nobiletin or a composition containing the same, the osteoclast differentiation inhibitor and oral preparation of the present embodiment contain various carriers, additives, and other medicinal agents within the scope of the effects of the present invention. Ingredients and the like may be included.

In addition, as for the form of the food and drink composition, it can be mixed with various food raw materials and additives, for example, in the form of biscuits, snacks, gums, chewable tablets, soft drinks, drinks, soups, jellies, candies, and the like. can do.

EXAMPLES Hereinafter, embodiments of the present invention will be described with reference to Examples, but the scope of the present invention is not limited by these.

<Example 1> Preparation of 4'-demethyl nobiletin-containing composition A composition containing 4'-demethyl nobiletin converted from nobiletin was prepared by fermenting ponkan pericarp aspergillus oryzae according to the method described in Japanese Patent No. 5667561. bottom.

That is, 10 kg of ponkan pericarp was chopped into small pieces and sterilized by steaming. Aspergillus awamori (manufactured by BIOC Co., Ltd.) was inoculated into the obtained ponkan pericarp so as to be distributed throughout. Then, aspergillus fermentation was performed aerobically for 5 days in a constant temperature room at 30°C to obtain an aspergillus fermented product.

60 L of water was added to 5 kg of the resulting fermented product of Aspergillus oryzae, and after hot water extraction at 100° C. for 1 hour, a hot water extract was obtained. The resulting extract was applied to a Diaion HP20 (porous synthetic adsorption resin column) equilibrated with water in advance, and after removing non-adsorbed components with 3 L of water, 2 L of 35% (v/v) ethanol was added. The component eluted at was removed. The components eluting with 2 L of 45% (v/v) ethanol were then collected. The collected material was further concentrated to dryness using an evaporator to obtain a 4'-demethyl nobiletin-containing composition (4'-demethyl nobiletin content: 19.1%).

<Example 2> Preparation of 4'-demethyl nobiletin isolate 2 g of the 4'-demethyl nobiletin-containing composition obtained in Example 1 was dissolved in 20% (v/v) methanol and subjected to ODS column chromatography. (30 g of Wakogel 50C18 was packed in a 30 cm long column with an inner diameter of 20 mm). Components eluted at 40% (v/v) methanol were removed to obtain components eluted at 60% (v/v) methanol.

Next, the obtained component was subjected to preparative TLC chromatography (silica gel 70PF ₂₅₄ plate Wako, film thickness 0.75 mm, manufactured by Wako Pure Chemical Industries) under the conditions of developing solvent hexane/ethanol = 7:3, and 4'-demethyl Fractions containing nobiletin were collected while checking with a UV lamp.

Then, the obtained fraction was subjected to a preparative HPLC column (TSK GEL ODS, manufactured by Tosoh Corporation, 4.6 mm x 25 cm), and pure 4'-derivative was purified by a mobile phase of 37% (v/v) acetonitrile. 20 mg of methyl nobiletin was isolated.

<Example 3> Investigation of Osteoclast Differentiation Inhibitory Action Using the 4'-demethyl nobiletin isolate obtained in Example 2 as a test substance, osteoclast differentiation inhibitory action was examined. RAW264.7 cells (mouse macrophage-like cells) were used as osteoclast differentiation cells.

Cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator at 37°C in the presence of 5% CO ₂ , and passaged every 3 to 4 days by treatment with 0.25% trypsin containing 0.1% EDTA. rice field.

RAW264.7 cells were seeded in a 96-well plate (1×10 ³ cells/well), and sRANKL (final concentration 300 ng/ml, manufactured by Wako Pure Chemical Industries) and 4′-demethyl nobiletin isolate (final concentration 5 μM) were added. was added and cultured. As a control, an equivalent amount of nobiletin (manufactured by Wako Pure Chemical Industries, Ltd.) was added in place of 4'-demethyl nobiletin.

Osteoclast-specific TRAP staining was performed after 5 days. That is, after removing the culture medium from each well and washing with PBS, the cells were fixed to the wells, stained with a tartaric acid-resistant acid phosphatase (TRAP) staining kit, and then microscopically observed. Red-staining (TRAP staining-positive) multinucleated cells were defined as osteoclasts, and the ratio of osteoclasts in all cells was used as an index of osteoclast differentiation. The inhibitory effect of the test substance on osteoclast differentiation was evaluated from the ratio of osteoclast differentiation with the addition of the test substance to no addition (negative control).

FIG. 1 is a micrograph showing the results of TRAP staining when sRANKL was not added and when sRANKL was added at 300 ng/ml. In FIG. 1, the left shows the results of the negative control (no addition), and the right shows the results of the addition of sRANKL at 300 ng/ml. As shown in Figure 1, many cells (TRAP-staining-positive multinucleated cells) that differentiated into osteoclasts were observed with the addition of sRANKL at 300 ng/ml. The inhibitory action was examined.

FIG. 2 is a microscopic image showing the osteoclast differentiation inhibitory effect of adding 4'-demethyl nobiletin isolate. In FIG. 2, the upper left shows the results of the negative control (no addition), the upper right shows the results of the addition of 300 ng/ml sRANKL, the lower left shows the results of sRANKL + 4'-demethyl nobiletin, and the lower right shows the results of sRANKL + nobiletin. In the sRANKL-added section (upper right of Fig. 2), many cells differentiated into osteoclasts (TRAP-staining positive multinucleated cells) were occupied. was not seen at all. From this, it was found that the addition of 4'-demethyl nobiletin exhibited a strong osteoclast differentiation inhibitory effect. In addition, in the group added with 5 μM nobiletin (bottom right of FIG. 2), a clear decrease in the number of TRAP staining-positive multinucleated cells was observed compared to the group added with sRANKL (upper right of FIG. 2). The degree of decrease was weaker than in Fig. 2, lower left). Therefore, nobiletin was also found to have an osteoclast differentiation inhibitory effect, but 4'-demethyl nobiletin was shown to have a clearly stronger osteoclast differentiation inhibitory effect.

Therefore, TRAP staining was performed with the final concentrations of 4'-demethyl nobiletin or nobiletin set to 5, 10, and 20 µM. By counting the number of TRAP-staining positive multinucleated cells (osteoclasts) by microscopic observation and calculating the ratio of osteoclasts in all cells as an index of osteoclast differentiation inhibitory action, osteoclast differentiation by the test substance The inhibitory action was evaluated. In addition, 4'-demethyltangeretin or tangeretin was used as a control. As for 4'-demethyltangeretin, a pure product obtained by purifying the fermented product of Aspergillus oryzae according to the method described in Japanese Patent No. 5667561 was used.

The results are shown in FIGS. 3A and 3B. FIG. 3A is a graph showing osteoclast differentiation inhibitory action by addition of 4'-demethyl nobiletin. In FIG. 3A, the bar graphs show, from the left, the results of the negative control (no addition), sRANKL added at 300 ng/ml, 4'-demethyl nobiletin added at 5, 10 and 20 μM, and nobiletin added at 5, 10 and 20 μM. In addition, ** indicates that there is a significant difference (p<0.01) from the nobiletin-added group at the same concentration. FIG. 3B is a graph showing osteoclast differentiation inhibitory action by addition of 4'-demethyltangeretine. In FIG. 3B, the bar graphs show, from the left, the results of the negative control (no addition), sRANKL added at 300 ng/ml, 4'-demethyltangeretin added at 5, 10 and 20 μM, and tangeretin added at 5, 10 and 20 μM. In addition, ** indicates that there is a significant difference (p < 0.01) with respect to the group added with tangeretin at the same concentration, and * indicates that there is a significant difference (p < 0.05) with respect to the group with the same concentration of tangeretin added. indicates In FIGS. 3A and 3B, the vertical axis indicates the ratio (%) of osteoclast differentiation, and the bar indicates the standard deviation.

In FIG. 3A, nobiletin exhibited a dose-dependent osteoclast differentiation inhibitory effect compared to the sRANKL-added group at a final concentration of 5 to 20 μM. However, in the 4'-demethyl nobiletin-added group, no TRAP staining-positive multinucleated cells were observed in the final concentration range of 5 to 20 µM, and a remarkably stronger osteoclast differentiation inhibitory effect was observed compared to nobiletin. On the other hand, in FIG. 3B, 4'-demethyltangeretin exhibited a relatively strong inhibitory effect on osteoclast differentiation compared to tangeretin, but there was no significant difference compared to nobiletin shown in FIG. 3A. Thus, the significant increase in the inhibitory effect on osteoclast differentiation by demethylation of nobiletin is completely different from the change by demethylation of tangeretin, a polymethoxyflavonoid derived from the same citrus peel component. It has been shown.

Furthermore, instead of the 4'-demethyl nobiletin isolate, the 4'-demethyl nobiletin-containing composition obtained in Example 1 (4'-demethyl nobiletin content: 19.1%) was added. In the same manner as above, the effect on osteoclast differentiation was examined.

The results are shown in FIG. FIG. 4 is a micrograph showing the osteoclast differentiation inhibitory action of a composition containing 4'-demethyl nobiletin. In FIG. 4, the upper left is the negative control (no addition), the upper right is the addition of 300 ng/ml sRANKL, the lower left is the addition of 10 μg/ml of the composition containing sRANKL+4′-demethylnobiletin, and the lower right is the composition containing 20 μg of sRANKL+4′-demethylnobiletin. /ml added, the results are shown. In FIG. 4, in the sRANKL-added group (upper right of FIG. 4), TRAP staining-positive multinucleated cells occupied many, while the composition containing 4′-demethyl nobiletin was added together with sRANKL at a final concentration of 10 μg/ml (lower left of FIG. 4). Alternatively, when 20 μg/ml (lower right of FIG. 4) was added, no TRAP-staining positive multinucleated cells were observed. From this, it was shown that the 4'-demethyl nobiletin-containing composition also has a strong osteoclast differentiation inhibitory effect similar to the isolate.

From the above examples, 4'-demethyl nobiletin was found to have a very strong osteoclast differentiation inhibitory effect. The effect was remarkable even compared with nobiletin.

<Example 4> Manufacture of internal medicine (tablet) Tablets having the formulation shown below were manufactured by a conventional method.
4'-demethyl nobiletin 20 mg
Whey Calcium 80 mg
70 mg of lactose
Microcrystalline cellulose 20 mg
5 mg of calcium carbonate
Sucrose ester 5 mg
200 mg total

<Example 5> Production of beverage A beverage having the following composition was produced by a conventional method.
Content of each component in 180 ml Composition containing 4'-demethyl nobiletin of Example 1 0.026 g
(5 mg as 4'-demethyl nobiletin)
5 g apple juice concentrate
2 grams of lactose
5 g fructose corn syrup
Acidulant (citric acid) 0.3 g
Sweetener (sucralose) 0.01 g
Preservative (sodium benzoate) 0.03 g
water residue

According to the present invention, pharmaceuticals, quasi-drugs, and food and beverage compositions containing 4'-demethyl nobiletin, a raw material that has been used extensively as an ingredient without fear of side effects, as an active ingredient, and that have osteoclast differentiation inhibitory activity. make it possible to provide Therefore, the present invention can be used for pharmaceuticals and quasi-drugs that are effective in treating osteoporosis and bone resorption diseases such as alveolar bone resorption in periodontal disease, and in food and drink compositions for preventing and improving such diseases. There is expected. Osteoporosis is often caused by aging, and its prevention and treatment take a long period of time. Under such circumstances, the present invention using raw materials derived from food materials is expected to be in great demand as a material for maintaining QOL in the present age of longevity society.

Claims (4)

An osteoclast differentiation inhibitor containing 4'-demethyl nobiletin as an active ingredient. A food and drink composition for suppressing osteoclast differentiation containing 4'-demethyl nobiletin as an active ingredient. An oral preparation for prevention or treatment of bone resorption diseases, containing 4'-demethyl nobiletin as an active ingredient. A food and drink composition for preventing or improving bone resorption diseases, containing 4'-demethyl nobiletin as an active ingredient.

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